Electrospray Ionization Mass Spectrometric Detection Research Articles

Methodology of analysis of very weak acids by isotachophoresis with electrospray-ionization mass-spectrometric detection: Anionic electrolyte systems for the medium-alkaline pH range

This work describes for the first time a functional electrolyte system setup for anionic isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection in the neutral to medium-alkaline pH range. So far no application was published on the analysis of very weak acids by anionic ITP-MS although there is a broad spectrum of potential analytes with pKa values in the range 5–10, where application of this technique promises interesting gains in both sensitivity and specificity. The problem so far was the lack of anionic ESI-compatible ITP systems in the mentioned pH range as all typical volatile anionic system components are fully ionized at neutral and alkaline pH and thus too fast to suit as terminators. We propose an original solution of the problem based on the combination of two ITP methods: (i) use of the hydroxyl ion as a natural and ESI-compatible terminator, and (ii) use of configurations based on moving-boundary ITP. The former method ensures effective stacking of analytes by an alkaline terminator of sufficiently low mobility and the latter offers increased flexibility for tuning of the separation window and selectivity according to actual needs. A theoretical description of the proposed model is presented and applied to the design of very simple functional electrolyte configurations. The properties of example systems are demonstrated by both computer simulation and experiments with a group of model analytes. Potential effects of carbon dioxide present in the solutions are demonstrated for particular systems. Experimental results confirm that the proposed methodology is well capable of performing sensitive and selective ITP-MS analyses of very weak acidic analytes (e.g. sulfonamides or chlorophenols).

Capillary moving-boundary isotachophoresis with electrospray ionization mass-spectrometric detection and hydrogen ion used as essential terminator: Methodology for sensitive analysis of hydroxyderivatives of s-triazine herbicides in waters

Capillary isotachophoresis (ITP) is an electrophoretic technique offering high sensitivity due to permanent stacking of the migrating analytes. Its combination with electrospray-ionization mass-spectrometric (ESI–MS) detection is limited by the narrow spectrum of ESI-compatible components but can be compensated by experienced system architecture. This work describes a methodology for sensitive analysis of hydroxyderivatives of s-triazine herbicides, based on implementation of the concepts of moving-boundary isotachophoresis and of H+ as essential terminating component into cationic ITP with ESI–MS detection. Theoretical description of such kind of system is given and equations for zone-related boundary mobilities are derived, resulting in a much more general definition of the effective mobility of the terminating H+ zone than used so far. Explicit equations allowing direct calculation for selected simple systems are derived. The presented theory allows prediction of stacking properties of particular systems and easy selection of suitable electrolyte setups. A simple ESI-compatible system composed of acetic acid and ammonium with H+ and ammonium as a mixed terminator was selected for the analysis of 2-hydroxyatrazine and 2-hydroxyterbutylazine, degradation products of s-triazine herbicides. The proposed method was tested with direct injection without any sample pretreatment and provided excellent linearity and high sensitivity with limits of detection below 100ng/L (0.5nM). Example analyses of unspiked and spiked drinking and river water are shown.

Chemical Composition, Antibacterial and Antifungal Activities of Crude Dittrichia viscosa (L.) Greuter Leaf Extracts.

The small amount of data regarding the antifungal activity of Dittrichia viscosa (L.) Greuter against dermatophytes, Malassezia spp. and Aspergillus spp., associated with the few comparative studies on the antimicrobial activity of methanolic, ethanolic, and butanolic extracts underpins the study herein presented. The total condensed tannin (TCT), phenol (TPC), flavonoid (TFC), and caffeoylquinic acid (CQC) content of methanol, butanol, and ethanol (80% and 100%) extracts of D. viscosa were assessed and their bactericidal and fungicidal activities were evaluated. The antibacterial, anti-Candida and anti-Malassezia activities were evaluated by using the disk diffusion method, whereas the anti-Microsporum canis and anti-Aspergillus fumigatus activities were assessed by studying the toxicity effect of the extracts on vegetative growth, sporulation and germination. The methanolic extract contained the highest TPC and CQC content. It contains several phytochemicals mainly caffeoylquinic acid derivatives as determined by liquid chromatography with photodiode array and electrospray ionisation mass spectrometric detection (LC/PDA/ESI-MS) analysis. All extracts showed an excellent inhibitory effect against bacteria and Candida spp., whereas methanolic extract exhibited the highest antifungal activities against Malassezia spp., M. canis and A. fumigatus strains. The results clearly showed that all extracts, in particular the methanolic extract, might be excellent antimicrobial drugs for treating infections that are life threatening (i.e., Malassezia) or infections that require mandatory treatments (i.e., M. canis or A. fumigatus).

Phenolic profiles of Lauraceae plant species endemic to Laurisilva forest: A chemotaxonomic survey

In this work, the phenolic composition of several trees endemic to Madeira archipelago (Portugal) was studied. Specifically, the leaves of the most relevant species of the Lauraceae family (Laurus novocanariensis, Apollonias barbujana, Ocotea foetens, and Persea indica) have been analyzed. The screening of the main phenolic compounds in their methanol extracts has been performed by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI–MSn), identifying or tentatively characterizing almost 100 compounds, including a high number of proanthocyanidins (A- and B-type), which have been reported to present remarkable health benefits. Thirty-four compounds have been quantified, observing total individual phenolic contents (TIPCs) between 18.43 and 88.99mgg−1 dry extract, with the lowest TIPC in O. foetens and the highest in A. barbujana.

Quantitative analysis of a multicomponent system for liquid chromatography–mass spectrometry determination of diosgenin, dioscin and protodioscin in plant extracts of Tribulus terrestris

An approach for the simultaneous determination of dioscin, protodioscin, and diosgenin in plant extracts by the liquid chromatography–mass spectrometry method with detection limits from 5 to 20 ng mL–1 is developed. Reversed-phase chromatography with the C18 sorbent followed by positive ion electrospray ionization and tandem mass spectrometric detection is used to analyze plant extracts. The external calibration and the quantitative analysis of multicomponent system are used to calculate the content based on peak areas in the chromatograms of the extracted ions. The difference between the results is shown to be less than 10%. The proposed approach is tested in the analysis of ethanolic and aqueous methanolic extracts of the aerial parts of Tribulus terrestris.

Evaluation of antioxidant potential, enzyme inhibition activity and phenolic profile of Lathyrus cicera and Lathyrus digitatus: Potential sources of bioactive compounds for the food industry

The genus Lathyrus has great importance in terms of food and agricultural areas. In this study, the in vitro antioxidant activity (phosphomolybdenum, DPPH, ABTS, FRAP, CUPRAC and metal chelating) and enzyme inhibitory activity evaluation (acetylcholinesterase, butyrylcholinesterase, α-amylase and α-glucosidase) of L. cicera and L. digitatus were investigated, as well as their phytochemical profiles. The screening of the main phytochemical compounds in aerial parts of L. cicera and L. digitatus was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn), observing that flavonoids represent the highest percentage of identified compounds, with abundance of tri- and tetra-glycosilated flavonoids, including acylated ones, especially in L. cicera. Generally, L. digitatus exhibited stronger antioxidant and enzyme inhibitory activities in correlation with its higher level of phenolics. The high number of phenolic compounds and the results of the antioxidant and enzyme assays suggest that these plants may be further used as sources of bioactive compounds, and for the preparation of new nutraceuticals.

A rapid UPLC-MS/MS assay for eicosanoids in human plasma: Application to evaluate niacin responsivity

A rapid and sensitive method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to simultaneously quantify hydroxyeicosatetraenoic (HETE), dihydroxyeicosatrienoic (DiHETrE), epoxyeicosatrienoic acid (EET), and prostaglandin metabolites of arachidonic acid in human plasma. Sample preparation consisted of solid phase extraction with Oasis HLB (30mg) cartridges for all metabolites. Separation of HETEs, EETs, and DiHETrEs was achieved on an Acquity UPLC BEH C18, 1.7µm (100×2.1mm) reversed-phase column (Waters Corp, Millford, MA) with negative electrospray ionization mass spectrometric detection. A second injection of the same extracted sample allowed for separation and assessment of prostaglandin metabolites under optimized UPLC-MS/MS conditions. Additionally, the endogenous levels of these metabolites in five different matrices were determined in order to select the optimal matrix for assay development. Human serum albumin was shown to have the least amount of endogenous metabolites, a recovery efficiency of 79−100% and a matrix effect of 71 – 100%. Linear calibration curves ranging from 0.416 to 66.67ng/ml were validated. Inter-assay and intra-assay variance was less than 15% at most concentrations. This method was successfully applied to quantify metabolite levels in plasma samples of healthy control subjects receiving niacin administration to evaluate the association between niacin administration and eicosanoid plasma level response.

Characterization and Quantification of Phenolic Compounds from Leaf of Agarista salicifolia

Agarista salicifolia is the wild tree and has been used as important medicinal plants in South Ethiopia since a long time ago. The aim of this work was to carry out a chemical analysis focusing on secondary metabolites, particularly phenolic compounds, which have several roles in the plant physiological processes and have demonstrated significant capacity in the prevention of human health diseases. Phenolic acids, flavonols, flavan-3-ols, and dihydrochalcones were characterized and quantified using ultra high-performance liquid chromatography coupled with diode array and electrospray ionization mass spectrometric detection (LC-MS), FTIR, and UV-Visible spectroscopy techniques. After extraction with ethanol, compounds were characterized and quantified based on retention time, molecular ions, and the comparison with reference compounds. Six flavan-3-ols, four phenolic acids, five flavonols, and two dihydrochalcones were identified and quantified in the extract. Chlorogenic (279.23 ± 28.75 µg/g of dried leaf) acid, Quercetin-3-O- rhaminoside (97.24 ± 4.07 µg/g of dried leaf), and epicatechin (304. 57 ± 20.88 µg/g of dried leaf), were the most abundant phenolic acid, flavonol, and flavanols, respectively. This is the first study on the phenolic composition of A. salicifolia, highlighting the importance of these natural products as a source of medicinal applications in pharmaceutical industries by herbal drug manufacturers.

Characterization of the Polyphenolic Profiles of Peel, Flesh and Leaves of Malus domestica Cultivars Using UHPLC-DAD-HESI-MSn

Qualitative and quantitative analyses of polyphenols extracted from 21 Malus domestica cultivars using ultra high performance liquid chromatography with diode array detection coupled to heated electrospray ionization mass spectrometric detection was performed for separation of 27 phenolic compounds on a reversed phase UHPLC column with an optimized gradient consisting of 1% formic acid in water and 1% formic acid in methanol within 20 minutes. According to retention times, UV maxima and mass spectra of the peaks in the chromatograms obtained from extracts of apple peel, flesh and leaves, the polypfienolic compounds were identified and quantified. Based on fragmentation patterns, 6 phenolic acids, 5 flavan-3-ols, 5 dihydrochalcones, 8 flavonols and 3 flavone derivatives were characterized in the studied samples. The method was then employed for analysis of the polyphenolic pattern of 21 apple cultivars, both commercial and autochthonous for the Macedonian region, as well as for monitoring the influence of long term storage on the polyphenolic content and composition of apple fruits and for comparison of polyphenolic profiles of apple cultivars during two years of harvesting. The obtained results revealed minor differences in the quality and major variation in the content of phenolic compounds in the flesh, peel and leaves in the studied apple cultivars that is attributed mainly to cultivar differences and meteorological factors.

Molecular profiling of naphthenic acids in technical mixtures and oil sands process-affected water using polar reversed-phase liquid chromatography-mass spectrometry.

In this work, a reversed-phase ultra-HPLC (UHPLC) ultrahigh resolution MS (UHRMS) method was evaluated for the comprehensive profiling of NAs containing two O atoms in each molecule (O2NAs; general formula Cn H2n + z O2 , where n is the number of carbon atoms and z represents hydrogen deficiency). Using a polar cyanopropyl-bonded phase column and negative-ion electrospray ionization mass spectrometric detection at 120,000 FWHM (m/z 400), 187 and 226 O2NA species were found in two naphthenic acid technical mixtures, and 424 and 198 species with molecular formulas corresponding to O2NAs were found in two oil sands process-affected water samples (one from a surface mining operation and the other from a steam-assisted gravity drainage operation), respectively. To our knowledge, these are the highest numbers of molecular compositions of O2NAs that have been profiled thus far in environmental samples. Assignments were based on accurate mass measurements (≤3 ppm) combined with rational molecular formula generation, correlation of chromatographic behavior of O2NA homologues with their elemental compositions, and confirmation with carboxyl group-specific chemical derivatization using 3-nitrophenylhydrazine. Application of this UHPLC-UHRMS method to the quantitation of O2NAs in the surface mining operation-derived water sample showed excellent linearity (R2 = 0.9999) with external calibration, a linear range of 256-fold in concentration, and quantitation accuracies of 64.9 and 69.4% at two "standard substance" spiking levels.

Characterization and quantification of flavonoid glycosides in the Prunus genus by UPLC-DAD-QTOF/MS.

Widely distributed in plants, flavonoids reduce the incidence of cancer and cardiovascular disease. In this study, flavonoid content and composition in members of the Prunus genus were evaluated using liquid chromatography with diode array and electrospray ionization mass spectrometric detection (UPLC-DAD-ESI/QTOF-MS). Flavonoids in plants of the Prunus genus include the basic structures of kaempferol, quercetin, and catechin, and exist as mono-, di-, or tri-glycoside compounds mono-acylated with acetic acid. A total of 23 individual flavonoids were isolated and confirmed, three of which appear to be newly identified compounds: quercetin 3-O-(2″-O-acetyl)neohesperidoside, quercetin 3-O-(4″-O-acetyl)rutinoside, and kaempferol 3-O-(4″-O-acetyl)rutinoside. Japanese apricot and Chinese plum contained the highest amounts of flavonoids in the Prunus genus. During the ripening stage of Japanese apricot, the total flavonol content was reduced, while the catechin content was increased.

Ulex europaeus: from noxious weed to source of valuable isoflavones and flavanones

The screening and quantification of the main phenolic compounds in leaves and flowers of Ulex europaeus (gorse) was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI–MSn) after ultrasound-assisted extraction with methanol. About 98% of compounds corresponded to flavonoids, distributed as flavonols, flavones, isoflavones and flavanones. Flavonols were mainly quercetin glucosides; most of the found flavones were apigenin derivatives and the isoflavone group was dominated by glycitin. The flavanone group was composed mainly of liquiritigenin derivatives, substances usually found in liquorice (Glycyrrhiza ssp) and associated with high pharmacological relevance; in Ulex they represent about 25% of total polyphenols content. Phenolic acids and saponins were also detected, as minor components. In vitro antioxidant activity (nitric oxide, superoxide assays, ABTS and DPPH assays) of leaves and flowers, and their inhibitory effects towards digestive enzymes related to carbohydrate metabolism (α-glucosidase and α-amylase) were also studied.

Comparative evaluation of nutritional compositions, antioxidant capacities, and phenolic compounds of red and green sessile joyweed (Alternanthera sessilis)

The nutritional compositions, antioxidant capacities, and phenolic compounds of Alternanthera sessilis red (ASR) and green (ASG) in water and ethanolic extracts were compared. Nutritional compositions were determined according to an AOAC method. Total phenolic content was evaluated by a colorimetric reduction using the Folin–Ciocalteu method. High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric detection was used to the study phenolic compounds. Antioxidant capacities were evaluated by five different methods. The results showed that ASR contained significantly (p < 0.05) higher protein, fat, carbohydrate and mineral contents compared to ASG. The ethanolic extracts of both ASR and ASG presented significantly greater antioxidant capacity than the water extracts at p < 0.05. The total phenolic content exhibited a positive correlation with the antioxidant capacities, which could be one of the critical contributors to the antioxidant activity of ASR and ASG.

Extraction and Characterization of Phenolic Compounds from Rose Hip (&lt;i&gt;Rosa canina&lt;/i&gt; L.) Using Liquid Chromatography Coupled with Electrospray Ionization - Mass Spectrometry

Wild berry are a rich of natural compounds which provide them high antioxidant potential. The compounds which provide them these proprieties are known to be vitamins, flavonoids, anthocyanins and phenolic acids. The aim of this study was to extract and characterize bioactive compounds from rose hip (Rosa canina L.) currently found in Romania. A qualitative high-performance liquid chromatography coupled with electrospray ionization mass spectrometric (ESI-MS) detection in positive ion mode has been used to identify phenolic compounds from rose hip crude extract. The chromatograms revealed the presence of a large number of compounds (19), identified and grouped as phenolic acids and flavones/ols, flavan-3-ols and also anthocyanins. Based on obtained results these berries can be highly recommended as part of our diet. Also this finding represents a contribution to the chemical characterization of phenolic profile of rose hip.

Separation and identification of oligomeric phenylethoxysiloxanols by liquid chromatography-electrospray ionization mass spectrometry

Liquid chromatography-electrospray ionization mass spectrometry has been applied to qualitative analysis of oligomeric phenylethoxysiloxanols, a class of organosilanols as active intermediates to polyhedral silsesquioxanes. The phenylethoxysiloxanol samples were prepared by controlled acid-catalyzed hydrolysis and condensation of phenyltriethoxysilane at various molar equivalents of water (r1) and characterized by standard spectroscopic techniques. Using a gradient binary water–methanol mobile phase, these reaction products were resolved on octadecylsiloxane silica stationary phase and subsequently identified by online electrospray ionization mass spectrometric detection. Results show that the reaction products are composed of a multitude of linear and monocyclic siloxanol oligomers with various numbers of silicon atoms and hydroxyl groups, depending upon the reaction conditions used. With the r1 value increasing from 0.5 to 2.0, the chain lengths of the oligomers increase slightly but the numbers of hydroxyl groups increase considerably, accompanying by structural evolution from chains to rings. Characterization of the retention behavior of these oligomers indicates that hydrophobic interactions of phenyl and ethoxy groups with the stationary phase are responsible for their retention in reversed-phase liquid chromatography.

HPLC-ESI-MSn characterization of phenolic compounds, terpenoid saponins, and other minor compounds in Bituminaria bituminosa

Bituminaria bituminosa is a wild legume that can endure drastic conditions, including contaminated and degraded soils. It has been traditionally used as feeding for livestock, and different uses in folk medicine are known. The chemical composition of leaves and flowers from B. bituminosa is presented for the first time. The screening of phytochemical compounds was carried out using high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). More than 40 compounds were identified or tentatively characterized. A high percentage of the detected compounds corresponded to glycosylated flavonoids, especially from apigenin, although phenolic acids, lignans, and saponins were also identified.

Determination of phytochemicals and antioxidant activity of methanol extracts obtained from the fruit and leaves of Tunisian Lycium intricatum Boiss.

A comparative analysis of methanol extracts from fruit and leaves of Lycium intricatum Boiss., a Solanaceous shrubbery with the potential to become a high-value crop, was performed by means of liquid chromatography with photodiode array and electrospray ionisation mass spectrometric detection (LC/PDA/ESI-MS). The total phenolic (TPC), anthocyanin (TAC) and flavonoid (TFC) contents as well as the antioxidant capacity measured by four complementary methods were performed for each sample.The results showed the tested extracts to be rich sources of phenolics; in leaves polyphenols and flavonoids dominate, while in fruit anthocyanins dominate. Nineteen phenolic compounds were detected and fifteen were identified or tentatively characterised based on Photodiode-array ultraviolet visible (PDA) UV–Vis spectra, ESI-MS spectrometric data and spiking experiments with authentic standards. Rutin and chlorogenic acid are the major constituents of the leaves and fruit, respectively. Results obtained in this study have revealed that leaves exhibit better performance in all antioxidant assays.From these results it has been shown that extracts of L. intricatum have great potential as a source of phenolics for natural health products.

Analysis of phenolic compounds in leaves from endemic trees from Madeira Island. A contribution to the chemotaxonomy of Laurisilva forest species

Phenolic compounds present high antioxidant activity and, therefore, health promoting effects, serving as a type of preventive medicine. Hence, research on the chemical composition of plants with potential antioxidant value is of high interest.Forest cleaning, thinning, and pruning are beneficial activities that help maintaining healthy forests. In addition, they can provide vegetal material as source of valuable bioactive compounds that can have health promoting effects. In this work, the phenolic composition of several trees native to Madeira Archipelago (Portugal) was studied. Specifically, the leaves from Olea europaea ssp. cerasiformis, Ilex perado ssp. perado, Clethra arborea, and Heberdenia excelsa have been analyzed. The screening of the main phenolic compounds from their methanolic extracts has been carried out using high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). This is the first report on the phenolic composition of these Madeira native species, and more than 100 compounds have been detected and identified or tentatively characterized.

Myrica faya: a new source of antioxidant phytochemicals.

Myrica faya is a fruit tree endemic of the Macaronesia (Azores, Madeira, and Canary Island), and its edible fruits are known as "amorinhos" (little loves), bright red to purple berries, used fresh and in jams and liquors. The phenolic composition and antioxidant capacity of leaves and berries from M. faya are presented here for the first time. The screening of phytochemical compounds was carried out using high-performance liquid chromatography with online UV and electrospray ionization mass spectrometric detection (HPLC-DAD-ESI-MS(n)). There were 55 compounds characterized, mostly galloyl esters of flavonoids and phenolic acids; 26 of the identified compounds (anthocyanins, isoflavonoids, lignans, terpenes, fatty acids, and phenylethanoids) have not been reported in Myrica genus so far. From the data presented here, it can be concluded that faya berries represent a rich source of cyanidin-3-glucoside, flavonoids, and vitamin C. In fact, higher antioxidant activity than that of the well-known Myrica rubra berries (Chinese bayberry) has been observed.

Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization, significantly reduces the matrix effects and provides a great analytical tool for the isolation of small molecules from human plasma.